Eukaryotic protein translation initiation factor 5A (eIF-5A) contains one residue of hypusine and appears to be the only cellular protein with this unique amino acid. Hypusine is produced post-translationally by transfer of the butylamine portion of the polyamine spermidine to a lysyl residue in the eIF-5A precursor to form deoxyhypusine followed by hydroxylation to form hypusine. The precise physiological role of the hypusine-containing protein eIF-5A is yet unknown. However, it is well established that hypusine is vital for eukaryotic cell proliferation. The strict conservation of the sequence of twelve amino acids surrounding the hypusine residue further emphasizes the importance of this residue. We investigated the basis for the specificity of hypusine synthesis with respect to the substrate protein using fragments of eIF-5A precursor protein as substrates for deoxyhypusine synthase, the first enzyme in hypusine biosynthesis. These were generated either by specific endoproteinases or by recombinant deletion subcloning. The results define the minimum domain of the eIF-5A precursor protein required for deoxyhypusine synthesis as Phe 30-Asp 80 and provide insight into the molecular interaction between eIF-5A precursor and deoxyhypusine synthase. In an effort to identify cellular proteins with which eIF-5A interacts to exert its biological activity, we have originated an affinity method for their isolation. Polyhistidine-tagged human eIF-5A precursor protein (His- tag-ec-eIF-5A) was produced by utilizing pET-24b vector. The deoxyhypusine-containing form was prepared by modification of His-tag-ec- eIF-5A in the deoxyhypusine synthase reaction. The his-tagged proteins bound to a Ni (II)-NTA-agarose column are being used as an affinity matrix to isolate and identify cellular proteins that specifically interact with eIF-5A. In a recent study from another laboratory, eIF-5A was reported to be a host cellular factor required for Rev function in the replication of HIV- 1. We are currently investigating the role of hypusine modification in the interaction of eIF-5A and Rev and the possible intervention of HIV-1 replication through inhibition of hypusine synthesis.